Our previous studies have shown that hydroxyurea (HU), in addition to its known effects on stimulating gamma-globin chain production, may increase beta-globin chain biosynthesis in some beta+-thalassemia patients with the IVS-2-654 C->T splicing mutation, in which a small amount of normally processed beta-globin mRNA is still produced. As a result, HU-treated patients showed an improvement in the effectiveness of erythropoiesis and alleviation of clinical symptoms. In order to further demonstrate the mechanisms of these changes, we used two-phase liquid human adult erythroid (hAE) cell culture procedure for growing erythroid precursors from two patients with the same IVS-2-654 mutation, coupled with serial RT-PCR mRNA quantitation measurement as well as microglobin chain biosynthetic assays to investigate globin gene expression patterns following HU treatment. The results showed that the level of normally spliced beta-globin mRNA (beta) as compared to the aberrantly spliced form (beta*) was significantly increased in the patients' cultured cells after HU treatment. Globin chain biosynthesis also showed an increase in beta- globin chain production. These results suggest that the increased level of normal beta-globin mRNA may result in part from an improvement in the splicing defect of the IVS-2-654 mutation. They are consistent with our previous findings in beta-thalassemia patients that HU may have a more general role in regulating globin gene expression. Our results are of significance both with respect to developing potential therapies for hemoglobin disorders and gaining insights into the regulation of globin expression.